Bioremediation Effect of Contaminated Soils with Copper and Lead on Sweet Basil Plants

INTRODUCTION

Aromatic and medicinal plants are an important source of national income and foreign currency in Egypt. Basil is one of the most important species for export among the medicinal and aromatic plants and it has a good reputation in the European countries. The area cultivated with basil in Egypt is about 4-5 thousand feddans, and the exports are more than 4000 tons per year (El-Sayed et al., 2003). The genus O. cimum comprises more than 150 species and is considered as one of the largest genera of the Lamiaceae family (Evans, 1996). Ocimum basilicum L. (sweet basil) is an annual herb, plants is widely used in food and oral care products; the essential oil of the plant is also used as perfumery   (Bauer et al., 1997) and  antimicrobial (Chiang, 2005). Environmental conditions and agricultural practices can significantly alter yield and chemical composition of sweet basil (Sifola and Barbieri, 2006).

Monitoring for toxic heavy metals in medicinal plants has become part of the quality control in the pharmaceutical industry as consumers demand products that are free from potentially harmful constituents (Chizzola et al., 2003). Phytoextraction potential of medicinal and aromatic plants grown in heavy metal polluted agricultural soils   is a subject of ongoing research (Kovačik et al., 2009; Stancheva et al., 2014).  In the rhizosphere, a synergism between various bacterial genera such as Bacillus, Pseudomonas, Arthrobacter and Rhizobium has been shown to promote plant growth of various plants such as sweet basil (Ocimum basilicum L.) (Hemavathi et al., 2006). Pseudomonas putida has the capacity to improve growth in plants (Rodriguez et al., 2014) particularly in sweet basil plants (Ordookhani et al., 2011). .Arbuscular mycorrhizal fungi (AMF) symbiosis is formed by approximately 80% of the vascular plant species in all terrestrial biomass (Smith et al., 2010) which improves plant productivity (Fedderman et al., 2010).  AM fungi can contribute plant growth, particularly in disturbed or metal polluted sites, by increasing plant access to relatively immobile minerals such as P, Zn, and Cu (Ryan and Angus 2003; Christie et al, 2004). In addition, they can improve soil structure (Gaur and Adholeya, 2004), stabilize metals in the soil, reduce their uptake, and thus decrease the risk of toxicity to plants growing in polluted substrates (González-Chávez et al., 2009).Thus, plants in symbiosis with AM fungi have the potential to take up heavy metals (HM) from an enlarged soil volume (Upadhyaya et al., 2010).

The aim of the present study was to examine the potency of two   isolates of AMF (Glomus intraradices), and Pseudomonas putida to reduce metal accumulation in plant organs, and to evaluate how inoculation effect of mycorrhization and essential oil composition of (Ocimum basilicum  L). cultivated in  Pb and Cu contaminated soil.

MATERIALS AND METHODS

Cultivated plant: Sweet Basil (Ocimum basilicum) seeds were obtained from Medicinal and Aromatic Plants Department, Agricultural Research Center, Ministry of Agriculture, Egypt. Seeds were germinated in seedling trail with 209 holes containing (1:1, v: v) peat moss and Sand. In case of mycorrhizal treatment (G3, G4), the inoculant was applied to the nursery at the rate of 10% added to the mixture. Seeds were sown in the nursery on April 16^th and the seedlings of 2-3 pairs of leaves were transplanted to the field at May 28^th with one plant per hill.

Soil initial state analysis: Some physical and chemical properties of surface layer (0-30 cm) of the experimental field   and heavy metals content (Pb and Cu) were determined according to (Page et al., 1982) and (Klute, 1986). The soil had the following physicochemical properties: clay loam in texture, pH (1:1 soil water suspension) 7.75 and 7.85, electrical conductivity (EC) 1.95 and 1.98 dSm^-1, available P (15, 14.7 mg kg^-1) soil, and the available Pb and Cu were (109, 100) and (90.3, 88.6) mg/ kg for the two seasons, respectively.

Experimental design: The field experiment was conducted in Abees Experimental Farm, Faculty of Agriculture, Saba Basha, 10^th Village, Alexandria University, Egypt during two seasons 2012 and 2013. The experiment was carried out in a randomized split – split plot (10.5m², 3.5 x 3m) design in 3 replicates. Phosphorus fertilizer (0%, 75%, and 100% of the recommended dose) was the main plot, and the two treatments un-inoculated and inoculated with Pseudomonas putida were randomly distributed in the sub- plot, while the three treatments with Glomus intraradices (control un-inoculated, G3 and G4) were arranged in the sub – sub plots.

Inoculation

Arbuscular Mycorrhizal Fungi: Two mycorrhizal isolates of Glomus intraradices were used in this experiment; isolate (G3) was isolated from the Experimental Station of Alexandria University at Abees (Aboul-Nasr, 1993), and isolate (G4) was obtained by Amykor Company, Germany. The inoculum consisted of expanded clay aggregates (2-4 mm in diameter, Leca) containing chlamydospores and fungus mycelium, which had been cullivated on Tagetes erecta L. (Aboul-Nasr, 2004). Inoculant was added at the rate of 7.0 g/per hill below basil seedling after transplanted. Control plants received the same amount of heat sterilized expanded clay.

Bacterial treatment: Pseudomonas putida obtained from Bio fertilization unit, Faculty of Agriculture, Ain Shams University was grown in liquid broth (LB) medium comprising of (g/L): tryptone, 10; yeast extract, 5; NaCl, 5. The pH of the medium was adjusted to 7.2–7.4 with 1 N HCl or 1 N NaoH and sterilized by autoclaving at 121 °C for 15 minutes. Incubation was carried at 35 °C for 3 days. When plants were transplanted to the field, 15 ml (1.3 ×10⁶ viable cells/ml) of bacterial suspension were added to each hill.

Mineral fertilizers: Three different rates (zero, 75% and 100%) of the recommended dose (400 kg/fed) of mono- calcium phosphate (15.5 % P₂O₅) was used in one does at seedling transplanted for each growing seasons. Ammonium nitrate (33.5% N) was added at the recommended dose150 kg/fed (50 kg N/ fed) after 30, 60, and 90 days of cultivation. Potassium sulfate (48% K₂O) was applied to all plots after 41 days of cultivation in the rate of 100 kg/fed.

The percentage of mycorrhizal colonization:The percentage of mycorrhization was estimated three times; (first, 4 weeks from germination to confirm the colonization before transfer to the field, second, 9 weeks old 1^st cut and third at 16 weeks old 2^nd cut). Root samples 1 cm were cleared with 10% KOH and stained with Trypan blue (0.05%) in lactophenol to observe under microscope (Koska and Gemma, 1989). Mycorrhizal colonization was estimated by determining the percentage of length of root segments containing AM fungal structure (arbuscules, vesicles, spores) according to Biermann and Linderman, (1981).

Vegetative parameters: Ten plantsamples per plot for each cut (1^st and 2^nd) were taken to determine the plant height (cm.) and herb fresh weight (g/plant). Five plants were air dried till constant weight and the average dry weight of root and shoot (g/plant) were calculated.

Chemical analysis

Phosphorus content: Plant powder (0.5g) was wet-digested with H₂SO₄ – H₂O₂ mixture (Lowther, 1980). Phosphorus was determined by the Vanadomolybdate yellow method (Jackson, 1967) using Millton Ray Spectronic 21 D.

Essential oil percentage: The essential oil percentage was determined in the air- dried herb according to British Pharmacopoeia (1963) by water distillation of 40 g of air dry herb for 1.5 – 2.0 hours, in order to extract the essential oil.

Essential oil analysis and its major components: The essential oils  were diluted in diethyl ether (20 :1, v:v) and analyzed with GC (HP 8644) with flame ionization detector (FID) on a fused silica 132 capillary column DB-5, 25 m in length, 0.32 mm i.d., and 0.5 mm film thickness.133 Helium was used as the carrier gas with a flow rate of 1.6 ml/min; the detector 134 temperature was 260 °C, the oven temperature was programmed to increase from 130  to 260 °C at a rate of 4 °C/min. The split injector was heated at 250 °C; the split 136 ratio was 15:1. Data were processed on a DP 800 integrator. The percentage of major constituents of stander (β-caryophellene, Linalool and Methyl chavicol) was estimated by measuring the peak area of the different compounds of the chromatogram according to (Heftman, 1967) and (Gunther and Joseph, 1978).Standards of the principal components which used as reference compounds for sweet basil were obtained from Ciba Gigi, NY, USA.

Total lead and copper analysis: Heavy metals content in shoots and roots were determined by digesting the dried plant of each plot according to (Lowther, 1980). The disgusted solutions were analyzed for lead and copper using the atomic absorption spectrophotometer (A Analyst 400) (Jackson, 1967). Available lead and copper remain in soil after cultivation were measured by atomic absorption spectrophotometer model (A.A. Spectrometry Thermo Elemental Type Solar 54/2001, ser No. GE 710728) using diethylene triamina penta acetic acid (DTPA) method as described by (Page et al., 1982).

Statistical analysis: Data were statistically analyzed by the procedure of variance to test the treatments effect on different measured parameters according to (Snedecor and Cochran, 1981). Data for the percentage of root length colonization % were analyzed using angular transformation (Steel and Torrie, 1982)

RESULTS

Mycorrhizal colonization percentage %  

Mycorrhization was determined three times during every growing season before plant transplanting (4 weeks old) and two times after transplanting (9 and 16 weeks) the colonization at the 4 weeks was more than 33.3%  that to confirm the colonization before transplanting. Root colonization by indigenous G. intraradices (G3 and G4) were low (Fig 1), but was enhanced by the inoculation with P. putida. Addition of P had no effect on mycorrhization in age 9 weeks but increased significantly after 16 weeks (Table1). However, the percent of root colonization was significantly higher in the basil inoculated with G. intraradices G3 and G4 than the non-inoculated plants. The AM root colonization in inoculated plants ranged from 32.99 to 55.10% and the native soil AM colonized roots were 7.72 % and 11.53 % at 9 and 16 weeks, respectively. The highest percent (55.10%) of colonization was noticed in plants with 16 weeks old with G3 and P. putida in the presence 75% of mineral phosphorus fertilizer. Generally the mycorrhizal root colonization hadn’t high value in this contaminated soil this could be attributed to the effect of heavy metals on the mycorrhizal spore germination, hyphal growth and branching.

(a) Plants with 9 weeks old

  (b) Plants with 16 weeks old

Fig (1). Effect of inoculating Ocimum basilicum L with Glomus intraradices (G3, G4) and Pseudomonas putida on mycorrhization after 9 and 16 weeks.

Table (1). A three-way ANOVA (LSD _0.05) showing the effect of P rates (P), P. putida (P.p) and Glomus intraradices (G.i) and their interactions.

   *: significant;ns: not significant

Vegetative parameters

Plant height (cm) and herb fresh weight (g/plant)

The results presented in Table (2) average values of cut one and cut two for the two seasons showed the growth responses of sweet basil grown in heavy metals polluted soil inoculated with Pseudomonas putida and AM fungi (G3 , G4) in the presence of different mineral phosphorus fertilizer rates. Data revealed that the plant height and herb fresh weight significantly increased in case of inoculated plants with both bacteria and mycorrhizal individually or in dual treatments, and the interaction between application of phosphorus and AM fungi had the same trend, additionally the interaction between bacteria and phosphorus or between bacteria and AMF had not affected, also the interaction between bacteria, AMF and phosphorus had the same trend except at the 2^nd cut (16 weeks old).   

Shoot and root dry weight (g/plant)

Sweet basil shoot and root dry weights (Table 3) responded significantly to phosphors application and AM inoculation, although the bacterial treatment had no significant impact in shoot dry weight in case of the second cut. Best results of shoots dry weight obtained with the addition of P_75% inoculated with P. putida and AM isolate G4 in the second cut. However the best results of roots dry weight (17.19 g/plant) obtained with addition P_100% and inoculation with bacteria and G4 in the 1^st cut. Additionally no interaction could be observed in shoots or roots dry weight between the factors P nutrition and inoculation with P. putida while, root dry weight significantly increased in case of the interaction between the factor of inoculation with P. putida and AMF or the interaction between P, P. putida and AMF except at 1^st cut for the latest interaction.

Phosphorus uptake (mg/g shoot) and oil content %

Data presented in Table (4) clearly show that the P uptake significantly increased, although the interaction between all factors had no impact. Application of phosphorus with different rates increased the phosphorus uptake of sweet basil plants, in the presence of inoculants (AM and bacteria). The highest P uptake observed in plants inoculated with G4 with bacteria in 75% of phosphorus fertilizer was (0.194 mg/g shoot). Although the highest percentage increase 268.8% was clear in G4 with bacteria. On the other hand the applied treatments improved the essential oil percentage compared with untreated plants.  AMF isolate G4 was the most effective treatment in increasing the oil content under contaminated soil its value was (0.47%), moreover the interaction had a significant differences between all factors. Table (4) showed that the percentage increase of oil content in plants inoculated with AM isolate G3 and G4 were (80 and 88%) , respectively more than the un-inoculated basil plants. No significant differences were observed between the duel inoculations with bacteria and phosphorus application and between mycorrhizae too.

Table (2). Effect of inoculated Ocimum  basilicum L. with Glomus intraradices and Pseudomonas putida  on plant height (cm) and herb fresh weight (g/plant) in lead and copper contaminated soil as an average of the two growing seasons 2012 and 2013

  Values are means± SE, n= 10 plants,*: significant; ns: not significant

Table (3). Effect of inoculated Ocimum basilicum L. with Glomus intraradices and Pseudomonas putida on shoot and root dry weight (g/plant) in lead and copper contaminated soil as an average of the two growing seasons 2012 and 2013

   Values are means± SE, n= 5 plant,*: significant; ns: not significant